Project IV: Computational Analysis of piRNA Biogenesis, Function, and Evolution Zhiping Weng, P. I. Project Summary piRNAs are the most recently discovered class of small silencing RNAs. They are primarily expressed in gonads, processed from long noncoding transcripts, and function to suppress expression of transposons and other repetitive elements. Failure of the piRNA pathway can activate transposons, compromise germline genome integrity, and cause sterility. Despite recent progress, many questions regarding the biogenesis and functions of piRNAs remain unanswered. What features distinguish piRNA-producing loci from the mRNAs that are not processed into piRNAs? The genomic structures of most piRNA loci are not precisely defined, hampering the identification of distinguishing sequence and structural features. What are the biological functions of mouse pachytene piRNAs? Since the majority of these piRNAs map to unique locations in the genome, it is unclear what they target or even if they have a function at all. How conserved are the components of the piRNA pathway in evolution? To address these questions, this project will use bioinformatic approaches to integrate high-throughput sequencing data with genetic and biochemical data. Much of the data require for this project are already available, and we will take advantage of the experimental data to be generated by the other projects of the PPG. Our results will inevitably lead to hypotheses, which will be tested experimentally via collaboration with the other projects. The project has three aims. Aim 1. To characterize the transcript structure of piRNA-producing genomic loci. Aim 2. To quantify the target recognition requirements of worm piRNAs and identify the functions of pachytene and mRNA-derived piRNAs in mouse. Aim 3. To study the evolution of the piRNA pathway. Relevance This project uses bioinformatic approaches to integrate high-throughput sequencing data with genetic and biochemical data to study the biogenesis, function and evolution of the piRNA pathways in mouse, fly and worm. Our results will provide insight into the mechanisms of germline development and ultimately benefit human fertility. 1